A major goal of the Human Genome Project is to provide researchers with an optimal infrastructure for finding and characterizing new genes. The availability of genetic and physical maps of the human genome may greatly accelerate the identification of human genes, including disease genes, and allow subsequent characterization of these genes. Once the genome maps and consensus sequences are obtained, the ability to assess individual variation may open the way to gene discovery and gene diagnosis. Such gene discovery programs may lead to new insights into the organization and functioning of the human genome and its role in the etiology of disease, providing new and highly accurate diagnostic and prognostic tests. Ultimately, the availability of filly characterized genes encoding a variety of functions may provide the raw materials for novel gene therapies and rational drug discovery/design. Other benefits may be recognized.
Rapid and accurate identification of DNA sequence heterogeneity has been recognized as being of major importance in disease management. Comprehensive testing for gene mutational differences can provide diagnostic and prognostic information, which, in the context of integrated relational databases, could offer the opportunity for individualized, more effective health care. Practical examples include current attempts to initiate pre-symptomatic testing programs by looking for mutations in genes predisposing to common diseases such as breast and colon cancer.
A recent estimate for single-nucleotide polymorphism (SNP) due to single-base substitution in the genome approximates 1 SNP/1000 bp. Other types of SNP involve insertion and deletion and are found to occur at ˜ 1/12 kb. Thus far, nucleotide sequencing remains the gold standard for accurate detection and identification of mutational differences. However, large-scale DNA sequencing to detect mutations is not efficient because of the low frequency of SNP. Furthermore, the high costs involved in sequencing have prompted the development of a large number of potentially more cost-effective, alternative, pre-screening techniques. These include single-stranded conformation polymorphism (SSCP) and SSCP-derived methods, chemical or enzymatic mismatch cleavage, denaturing gradient gel electrophoresis (DGGE), matrix-assisted laser desorption/ionization mass spectrometry, 5′nuclease assay, single nucleotide primer extension, and chip-based oligonucleotide arrays, among others.
Two-dimensional (2-D) gel electrophoresis is a commonly used technique for separating proteins based on molecular weight and isoelectric point. This technique is also used for separating DNA molecules based on size and base-pair sequence for detecting mutations or SNPs. The 2-D format for DNA separation increases the number of target fragments that can be analyzed simultaneously.
2-D DNA gel electrophoresis has been used to two-dimensionally resolve the entire E. coli genome and detect differences. DNA fragments can be resolved in two dimensions based on their differences in size and sequence. Sequence-dependent separation is typically achieved in the second dimension using DGGE. Apart from nucleotide sequencing, DGGE is the only known method which offers virtually 100% theoretical sensitivity for mutation detection. Provided the sequence of the fragment of interest is known, DGGE can be simulated on the basis of the melting theory using a computer algorithm. By attaching a GC-rich fragment to one of the PCR (Polymerase Chain Reaction) primers, the target fragment can be designed so that it will always be the lowest melting domain, providing absolute sensitivity to all kinds of mutations.
It is known to combine 2-D DNA gel electrophoresis with extensive PCR multiplexing to produce a high resolution system known as a two-dimensional gene scanning (TDGS) system. TDGS systems can be used for detecting mutational variants in multiple genes in parallel. The resolving power of TDGS has been demonstrated for several large human disease genes, including CFTR (cystic fibrosis transmembrane regulator gene), RB1 (retinoblastoma tumor suppressor gene), MLH1 (MutL protein homolog 1), TP53 (p53 tumor suppressor gene), BRCA1 (breast and ovarian cancer susceptibility gene 1), and TSC1 (tuberous sclerosis complex gene 1), as well as for a part of the mitochondrial genome.
To be suitable for true large-scale analysis, including for example, analysis of essentially all human genes in population-based studies, a mutation scanning system should not only be accurate but also capable of operating at a high throughput in a cost-effective manner. At present, 2-D DNA gel electrophoresis is relatively cost-effective in comparison with other mutation detection techniques. However, TDGS suffers from the fact that it is not a high-throughput platform for large-scale DNA analysis. Despite the selectivity and sensitivity of conventional 2-D DNA analysis, this technique as practiced today is a collection of manually intensive and time-consuming tasks, prone to irreproducibility and poor quantitative accuracy.
Microfluidic systems generally are known and are convenient for performing high-throughput bioassays and bioanalyses. One problem with existing systems is the materials and fabrication procedures used in existing commercial microfluidic devices. Currently, the majority of devices are made from glass or silicon. These materials are often chosen, not because of their suitability for the applications at hand, but rather because the technology is readily transferable from established procedures. A limitation with glass or silicon-based microfluidic devices is the high cost of fabrication and the brittleness of the material.
Separations by DGGE are based on the fact that the electrophoretic mobility of a partially melted DNA molecule is greatly reduced compared to an unmelted molecule. When a mixture of molecules, differing by single base changes, is separated by electrophoresis under partially denaturing conditions, they display different states of equilibrium between the unmelted DNA fragment and the partially melted form. The fraction of time spent by the DNA molecules in the slower, partially melted form varies among specific sequences. Less stable species move more slowly than the more stable ones in an electric field, resulting in efficient separation.
The generation of a temperature gradient in a capillary via ohmic heat produced by a voltage ramp over time is known, as is the use of DGGE in capillary electrophoresis. While these results have some favorable aspects, constructing the gradients is not quite straightforward, particularly for the development of multiple-capillary arrays. Others have demonstrated a 96-capillary array electrophoresis system for screening SNP by surrounding the capillaries with thermal conductive paste and controlling the temporal temperature gradient through the use of an external heating plate. Various drawbacks exist with these approaches.
Another problem with microfluidic devices for 2-D DNA gel electrophoresis is the lack of convenient, effective methodology to transfer DNA molecules from a first dimension to a second dimension after separation of molecules in the first dimension. Microfluidic devices for 2-D DNA gel electrophoresis also suffers from the lack of a convenient method or device for high throughput and high resolution second dimension separation. Current approaches using DGGE or other currently available gel based methods for this sequence-dependent separation in microfluidic devices have limitations in handling for high throughput purposes.
These and other drawbacks exist with known systems and methods.